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Limb development in the Pacific giant
salamanders, Dicamptodon (Amphibia,
Caudata, Dicamptodontidae)
David B. Wake and Neil Shubin
Abstract: The larvae of Pacific giant salamanders, Dicamptodon (Dicamptodontidae), develop in streams, display
precocious limb development, and possess a full complement of digits at hatching. Limb morphogenesis was studied
using cleared whole mounts stained with Alcian Blue or by immunohistochemical methods to reveal stages of limb
development. Limb morphogenesis in these salamanders conforms to the general caudate pattern, i.e., preaxial
dominance and precocious development of an initially isolated digital arch, despite some differences in detail from
other salamander taxa. These comparisons support the hypothesis that a fundamental ground plan characterizes limb
development in salamanders which transcends adaptive modifications related to ecology and life-history evolution. This
ground plan differs from that which characterizes frogs and amniotes, which shows postaxial dominance and a digital
arch that arises from basal elements.
Résumé : Les larves des salamandres géantes du Pacifique, Dicamptodon (Dicamptodontidae), se développent dans les
ruisseaux, leurs membres apparaissent tôt et elles possèdent déjà tous leurs doigts à l’éclosion. La morphogenèse des
membres a été étudiée par examen de montages d’animaux entiers colorés au bleu Alcian ou par des méthodes
immunohistochimiques capables de montrer les stades du développement. En dépit de petites différences avec d’autres
taxons, la morphogenèse des membres chez ces salamandres est typique de celle qui prévaut chez les Caudata, soit la
dominance préaxiale et le développement précoce d’un arc digital d’abord isolé. Ces comparaisons confirment
l’hypothèse de l’existence d’un plan de base de développement des membres chez les salamandres, plan qui transcende
les modifications évolutives reliées à l’écologie ou à l’évolution de la démographie. Ce plan de base diffère de celui
qui caractérise les grenouilles et les amniotes dans lequel il y a dominance postaxiale et présence d’un arc digital
formé à partir d’éléments de base.
[Traduit par la Rédaction]
Wake and Shubin
Limb development in salamanders differs from that in all
other tetrapods in several important respects (Shubin and
Alberch 1986; Shubin and Wake 1996). However, details of
limb development have been studied only in species that
have pond-type larvae and in which limb development proceeds slowly, mainly taking place after hatching as the freeliving larvae move about (Shubin and Alberch 1986; Blanco
and Alberch 1992; Vorobyeva and Hinchliffe 1996). Thus,
limbs are moved as they are developing. Limb development
in anurans having free-living larvae is much delayed relative
to that in salamanders, and limb development in amniotes
takes place mainly within the egg or in utero; in neither instance are limbs functional as they are developing. Salamanders display a variety of life-history and development modes
(Duellman and Trueb 1986). Such variation offers the possi-
Received February 9, 1998. Accepted June 23, 1998.
D.B. Wake.1 Museum of Vertebrate Zoology, University of
California, Berkeley, CA 94720-3160, U.S.A.
N. Shubin. Department of Biology, University of
Pennsylvania, Philadelphia, PA 19104-6017, U.S.A.
1
Author to whom all correspondence should be addressed
(e-mail: [email protected]).
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2066
bility of examining the relative impact of history (phylogeny) and ecology (and by inference, adaptation) on the evolution of limb morphogenetic patterns. In a recent study,2
limb development was described in three salamanders of the
family Plethodontidae that undergo intracapsular limb development, one a species with precocious development of
stream-adapted larvae and the other two with direct development and no larval stage. Despite considerable divergence
from the pattern of limb development in pond larvae, the
major differences that distinguish salamander limb development from that in other tetrapods (Shubin and Alberch 1986)
remain. In this paper, we report results of a study of limb development in a member of a family, Dicamptodontidae, that
includes species which are the largest terrestrial salamanders. These salamanders have stream-type larvae rather than
the pond-type larvae that characterize most members of the
sister taxon, the Ambystomatidae. No studies of early development in dicamptodontids have been published previously.
The Pacific giant salamanders, Dicamptodon (Dicamptodontidae), are endemic to the northwestern United States and
southwestern British Columbia, Canada. There are four spe2
S.B. Marks, D.B. Wake, and N. Shubin. Limb development and
evolution in the salamander genera Desmognathus and
Bolitoglossa: separating hypotheses of ancestry, function and
life history. Submitted for publication.
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Fig. 1. Entire embryo of Dicamptodon tenebrosus, representing
day 0 of our sequence of limb development, showing the relative
size of the huge encapsulated embryo (12.2 mm SVL) stained
with antibodies to type II collagen. There is a moderately strong
anterior–posterior gradient of development. Staining of the
hyobranchial apparatus shows that the system is completely
formed. Paired neural pedicel rudiments are present for the
anterior vertebrae. Rudiments of the humerus, radius, and ulna
are seen in the developing forelimb. Scale bar = 2 mm.
cies, all having long-lived larvae (at least 2 years and sometimes more, with individuals that remain in a permanently
larval state documented in two species) that are of the
stream type (i.e., hatch at a relatively advanced stage of development with reduced fins and gills). Eggs are laid under
large rocks in streams and are rarely found (Nussbaum 1969;
Jones et al. 1990), so we took advantage of an unusual discovery to study the development of Pacific giant salamanders.
Our data are derived from two clutches of eggs of Dicamptodon
tenebrosus discovered in a small stream south of Yachats, Lane
County, Oregon, in 1991. The eggs were raised in the laboratory,
and observations of the resulting embryos are reported in this
paper. The two clutches were at advanced stages of development
when collected. They were transferred to the laboratory and maintained at 14°C until hatching. Every few days, two or three eggs
were preserved in 10% neutral buffered formalin or Dent’s solution
(80% methanol, 20% dimethylsulfoxide). Details of development and
a partial table of developmental stages will be published separately.3 One of the clutches (I) produced most of the specimens reported here. After close comparison of the preserved stages, clutch
II was estimated to be approximately 11 days older than clutch I.3
Selected embryos were fixed and then either cleared and stained
with Alcian Blue (for chondrification centers, four specimens) or
used for immunohistochemical studies. The latter specimens were
stained with antibodies for type II collagen to show the developing
skeleton. Successful stainings with type II collagen were obtained
for seven specimens ranging in size from 11.4 to 15.9 mm (snout
to posterior angle of vent, or snout–vent length (SVL)), and these
proved useful in visualizing early stages of morphogenesis. This
size range, which represents approximately 27 days, encompasses
limb development from a stage at which two digits are present on
the forelimb and none on the hind limb to one at which all four
digits are present on the forelimb but only two on the hind limb.
This series extends from late embryonic to early hatchling stages
(hatching occurs at about 13.4 mm SVL). Time from fertilization
3 R.S.
2059
to hatching is not known. The first day on which embryos were
preserved (August 21, 1991) is taken as day 0. This stage of development is approximately equivalent to stage 44 in the normal table
for Ambystoma maculatum of Harrison (1969) as far as the forelimb is concerned, but not for the hind limb, which is not present
until much later in development in A. maculatum. Comparisons
were made with specimens studied by Shubin and Alberch (1986)
and Marks et al. (see footnote 2) as well as with materials in the
collection of the Museum of Vertebrate Zoology (MVZ) at the
University of California. All specimens reported here are in the
collection of MVZ. Terminology follows Shubin et al. (1995).
Although we were limited by the small samples available,
we obtained critical information on limb development. The
limbs of Dicamptodon are more fully developed at hatching
than those of species that develop in ponds (i.e., Ambystoma,
Triturus, Salamandrella). At hatching, elements of the forelimb are completely differentiated, although not yet fully ossified. The hind limb contains at least two digits at hatching,
but its development continues to lag behind that of the forelimbs. This lag between the differentiation of the forelimbs
and hind limbs is not nearly as great as that seen in species
with pond-type larvae (e.g., A. maculatum; Harrison 1969).
The earliest stage of limb development available to us is
in an individual of 12.2 mm SVL, preserved on day 0 and
stained for collagen II (Fig. 1). Externally, two digits are visible in the forelimb, while no external differentiation of the
hind limb is discerned. Internally, the humerus, radius, and
ulna form a deeply stained and continuous Y-shaped precartilaginous condensation. The radius is stained and is
larger than the ulna. The only other precartilaginous
Anlage that is evident lies distally and is discontinuous
from the radius and ulna. This weakly stained region contains the Anlagen of the basal commune and digits 1 and 2.
Digit 2 is distinct, but digit 1 is only weakly indicated.
There is no internal staining in the hind-limb bud.
A specimen preserved on day 3 is smaller than the first
specimen (11.4 mm SVL) but is more highly differentiated.
Two digits are present externally in the forelimb (Fig. 2A),
but the hind limb remains undifferentiated. The humerus,
radius, and ulna are more deeply stained in this collagen II
preparation than in the preparation from day 0. Development
of the radius continues to be significantly advanced relative
to the ulna (Fig. 2A). Two columns of stained matrix extend
distally from the ulna. The lateral (postaxial) branch continues distally to merge with the Anlage of the third metacarpal. The medial (preaxial) branch of the ulna approaches the
basale commune distally but does not connect with it. The
basale commune and metacarpals 1 and 2 form a deeply
stained and continuous mass in which digit 2 remains the
dominant digit. Darkly stained extracellular matrix extends
postaxially from the basal commune to the rudiment of the
third distal carpal and metacarpal. A preaxial column extends distally from the radius; independent foci for radiale
and element y are not yet present. The hind limb continues
to lag behind the forelimb in development. The femur is
Beroukhim and D.B. Wake. Embryonic development in the northern Pacific giant salamander, Dicamptodon tenebrosus. In preparation.
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stained, but otherwise, there is only a slight darkening in the
vicinity of the future basale commune.
A collagen II preparation representing day 14 measures
12.0 mm SVL and shows a more advanced stage of limb development (Fig. 2B). The distal manus now contains three
distinct digits. The first two digits each contain the Anlagen
of a metacarpal and two phalanges. The basale commune is
a discrete focus, continuous with distal carpal 3 postaxially.
From distal carpal 3, a large and well-stained rudiment of
the third digit extends distally. A gap (i.e., unstained matrix)
separates the ulnare from the third distal carpal. The
intermedium and centrale lie within an undifferentiated column that is only weakly connected to the medial part of the
fully formed ulna. A rod of stained matrix extends from the
radius and contains the undifferentiated rudiments of both
the radiale and element y.
The hind limb of the day 14 specimen consists of a small
pad that only weakly shows the tips of digits 1 and 2
(Fig. 3A). The hind-limb preparation from day 14 is generally similar to that of the forelimb on day 3. The basale
commune and the rudiments of digits 1 and 2 are joined together in a continuous undifferentiated mass with the tips of
the digits widely separated from each other. A rudimentary
digital arch extends posteriorly from the basale commune toward the fibulare as a zone of stained matrix, and a second
medial zone of staining extends between these two regions,
forming a more or less continuous ring surrounding a zone
of unstained matrix. The undifferentiated rudiments of the
centrale and intermedium are contained within the medial
zone of staining extending from the fibula. Preaxially, there
is a discrete fibula from which a lightly stained rudiment extends distally. This preaxial column is well separated from
the ring and from the basale commune.
A collagen II preparation of a specimen preserved on day
22 reveals all four digits of the forelimb (Fig. 2C). Two phalanges are evident in digits 1 and 2, and the second phalanx
in the digit 2 is attenuated and elongated. Only a single phalanx is present in digit 3. The fourth digit is differentiated
but continuous with the rudiment of distal carpal 4. The digital arch is complete and distal carpal 4 is separated by a
zone of weak staining from the fibulare. The radiale and
ulnare are well formed and there is a distinct element y. The
preaxial column remains well differentiated from both the
medial series and the basale commune. The rudiment of the
intermedium is deeply stained but remains continuous with
an expanded rudiment of the centrale. The centrale has two
foci, one of which extends postaxially toward distal carpal 4
(Fig. 2C).
The hind limb of the preparation from day 22 now contains three digital rudiments (Fig. 3B), but these are barely
evident externally. A deeply stained basale commune is continuous with distal tarsal 3. The first two digits remain as
single elements that show a pattern of weak proximodistal
differentiation. Digit 3 is represented by a lightly stained rod
that is continuous with distal tarsal 3. The femur, tibia, and
fibula are all separate elements. The most prominent feature
in the proximal tarsus is a heavily stained column of matrix
that contains the future intermedium and centrale. Proximally this column is continuous with the tibia and fades distally as it reaches the basale commune. The postaxial branch
Can. J. Zool. Vol. 76, 1998
of the fibulare is weakly stained and poorly bounded; it represents the future fibulare.
A cleared and stained preparation (MVZ 215981, not
shown) from the second clutch appears to represent a later
stage of development, and we estimate that it represents
approximately a day 24 stage in the first clutch. The differences in resolution between immunohistological and cartilagestained preparations are vividly demonstrated by this specimen. Far less detail is evident than in the day 22 specimen
that was stained by immunohistochemistry. In the day 24 example the hind limb shows staining only in the femur. Much
more staining is present in the forelimb, where the humerus,
radius, and ulna are apparent. The ulna is considerably
smaller than the radius and is bifurcated distally. The ulnare
is separate from the ulna. The intermedium is the largest of
the three basal units of the manus, and it remains in continuity with the ulna. A distinct, distally pointed radiale is about
the same size as the ulnare and is separated from the radius.
There is a large unstained gap across the center of the manus
between the basale commune and the weakly differentiated
Anlagen of the centrale. The ulnare and radiale are both separated from the distal elements by a gap of unstained matrix.
The basal commune has the shape of a deep crescent with
each point connected to a metacarpal representing each of
the first two digits. Both of these digits have two distinct
phalanges, and in each case the second is elongate and
sharply pointed distally. A small, weakly stained element
postaxial to the basale commune represents distal carpal 3.
Extending distally from it is a small, weakly stained rudiment of the third digit.
A specimen from the second clutch (15.6 mm SVL) that is
immunologically stained (collagen II) represents the next
stage of limb morphogenesis. Using the 11-day correction
factor, this embryo is expected to be at a stage approximately
equivalent to day 31 of the first clutch. Limb morphogenesis
is somewhat less advanced than in a slightly larger specimen
from the first clutch that is at day 29 (see below). There has
been relatively little change from the day 22 embryo. No
more phalanges are present, but the main carpus is somewhat more consolidated and the elements are denser. The
centrale and intermedium remain continuous in a relatively
massive condensation, in contrast with what is seen in the
cartilage preparation from day 24. The ulnare now contacts
distal carpal 4, which is larger than in the earlier preparations, and this carpal is continuous with the rudiment of the
fourth digit. The first two digits are larger than previously,
and the phalanges are thicker and more compact. More significant changes are evident in the hind limb (Fig. 3C).
Three digits are represented, the third containing only a single element representing mainly the rudiment of the metatarsal. The first and second digits have one phalanx on one
side, and there is a second phalanx in the second digit on the
other side. The basal commune is distinct and separated
from all other elements. Distal tarsal 3 is also distinct. A
weakly stained digital arch bridges the gap between the distinct fibulare, which is well separated postaxially from the
fibula. Proximally, a large medial column is continuous with
the medial side of the distal end of the fibula. This column is
weakly differentiated into an intermedium and a centrale, the
latter being, in turn, well separated from the basale com© 1998 NRC Canada
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Fig. 2. Forelimb development of D. tenebrosus visualized with immunohistochemistry (stained with antibodies to type II collagen).
(A) Right forelimb of an embryo, day 3. The humerus (H), radius (R), and ulna (U) are well stained. The basale commune (bc) and
the bases of digits 1 (I) and 2 are joined, and the digital arch extends posteriorly and bifurcates to connect to the base of digit 3 (III).
A ring of staining extends from the ulnare (u) mediodistally in the direction of the basale commune and posterodistally in the direction
of the digital arch and surrounds an unstained area. The medial portion represents the rudiment of the intermedium (i). r, radiale. Scale
bar = 0.8 mm. (B) At day 14, the manus now contains discrete elements: radiale, intermedium–centrale, and ulnare from preaxial to
postaxial and basal, basale commune, digit 1 (containing metacarpal and phalanges 1 and 2), digit 2 (containing metacarpal and
phalanges 1 and 2, the latter very weakly stained), and digit 3 (consisting solely of an undifferentiated rudiment) distally. Scale bar =
0.12 mm. (C) At day 22 of limb development, four digits are now represented. The digital arch is complete and the condensations of
distal carpals 3 and 4 are evident. A complex of intermedium and centrale elements forms a medial condensation. Both radiale and
ulnare are distinct, as is element y. Distal carpal 3 is separate from digit 3 but remains connected to distal carpal 4. Scale bar =
0.12 mm. (D) The day 29 forelimb is more fully differentiated and the digits are longer and stouter. Scale bar = 0.16 mm.
mune. There is also a distinct preaxial column extending
from the distal end of the tibia. This column is weakly differentiated into the rudiments of the tibiale and the distally
pointed element y.
The latest stage of development in our series is a collagen
II preparation of a day 29 stage of limb development
(15.7 mm SVL). Although morphogenesis of the forelimb is
well advanced (Fig. 2D), the medial column containing the
rudiments of the centrale and intermedium remains consolidated into a mass that contains three main concentrations.
One of these foci (the intermedium) lies basally, while the
other two lie distally (these appear to be separate rudiments
of the centrale). Distal carpals 3 and 4 and the rudiment of
the metacarpal of the fourth digit are distinct. There are two
distinct phalanges in both the first and second digits, and the
terminal phalanx of the second gives evidence of further regional differentiation. Four centers of differentiation are
present in the third digit, but only the metacarpal and the
first phalanx are distinct. The preaxial column is differentiated into a radiale and element y. The postaxial column, consisting only of the ulnare, is separated from distal carpal 4.
Four digits are evident in the hind limb at day 29 (Fig. 3D),
although the fourth is weakly stained. The third and fourth
are undifferentiated regionally and are in continuity with
their associated distal tarsals. Each of these digits contains a
distinct metatarsal and two phalanges. The basale commune
is distinct and separate from other elements. There are three
distinct columns at the base of the tarsus. The preaxial column is differentiated into a tibiale and a weakly stained and
distally pointed element y. The intermedium and centrale are
distinct elements. The centrale appears to consist of two
foci, a preaxial focus and a more postaxial rudiment that
may represent element m (see Shubin et al. 1995). The
postaxial column consists of a deeply stained fibulare that
appears to become continuous distally with a weakly stained
digital arch extending postaxially and proximally from the
fourth distal carpal.
Limbs of adult Dicamptodon ensatus (a closely related
species) were studied in cleared and stained preparations
(Fig. 4). There are four digits and five toes, the standard pattern for salamanders, with phalangeal formulas of 2–3–2–2
from preaxial to postaxial in the manus and 2–2–3–3–2 in
the pes. The phalanges and metapodial elements are ossified,
but with large cartilaginous ends (save for the terminal phalanges, which are finished in bone). The mesopodial elements remain cartilaginous, but in some large individuals
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Can. J. Zool. Vol. 76, 1998
Fig. 3. Hind-limb development of D. tenebrosus visualized with immunohistochemistry (stained with antibodies to type II collagen).
(A) At day 14 of limb development, rudiments of the first two digits are present. The femur, tibia, and fibula are all stained, and a
weak ring of staining extends from the tibia to the condensation that marks the site of the basal commune, the rudiments of the digits,
and the digital arch. Scale bar = 0.15 mm. (B) Day 22 hind limbs show three digits. A basale commune and rudimentary digital arch
are evident. The femur, tibia, and fibula are all present, and the tibial rudiment bifurcates distally into a medial and a postaxial
column. Scale bar = 0.16 mm. (C) This specimen is estimated to be equivalent to specimens of day 31 of the clutch that produced the
other specimens in this series. Rudiments of three digits are present. The tibia and fibula are well stained. A medial column of
condensation is continuous with the distal, medial end of the tibia. This column shows weak differentiation into rudiments of the
intermedium and the centrale, but the centrale falls short of the distinct basale commune. The fibulare is small and isolated distally
from the fibula in the postaxial region. A preaxial column is weakly differentiated into rudiments of the tibiale and element y. Scale
bar = 0.24 mm. (D) At day 29 of limb development, there are now four digits (I–IV), although the fourth is only weakly stained. Two
phalanges are present in the first two digits, and the metacarpals are distinct, but only a single rudiment is present in digits 3 and 4
and it is continuous with the associated distal tarsal (3 and 4) in each instance. The basale commune (bc) is well formed and discrete.
An anterior column is differentiated into a tibiale (t) and element y (y). The medial column is relatively massive and only weakly
differentiated regionally into an intermedium (i) and centrale (c). The postaxial column contains a well-differentiated fibulare (fi) that
is in contact with the developing digital arch distally. The femur (F), tibia (T), and fibula (f) are all deeply stained. Scale bar =
0.12 mm.
they become mineralized to a varying extent. The number
and arrangement of the mesopodials is in the typical pattern
of derived salamander families such as the Ambystomatidae
and Salamandridae (Shubin and Wake 1996). In the manus
there are the three basal elements, the preaxial radiale, the
medial intermedium, and the postaxial ulnare. Element y is
present distal to the radiale. There is a single large centrale
in the middle of the manus, distal to the intermedium. The
basale commune and two distal carpals complete the mesopodium. In the pes there are again three basal elements, the
preaxial tibiale, the medial intermedium, and the postaxial
fibulare. Element y is present distal to the tibiale. There is a
single large centrale in the middle of the pes, distal to the
intermedium. The basale commune and three distal tarsals
complete the mesopodium.
Dicamptodon has a generalized morphology, and three of
the four species metamorphose as fully as any other salamander with a metamorphic life history. Metamorphosed
dicamptodontids are large, well-ossified organisms with
large, well-developed limbs. These traits make them appropriate for comparing with related taxa, especially Paleozoic
amphibians. There are features of Dicamptodon that make
them attractive for studies of development and evolution, including an extended period of intracapsular embryonic development. They have stream-adapted larvae, characterized
by advanced development at hatching (the limbs are relatively well developed at this point), perhaps associated with
survival in rapidly flowing streams where they hatch. In
these respects, dicamptodontids differ from the three species
of salamanders in which limb development has been studied
most thoroughly to date, Ambystoma mexicanum (Shubin
and Alberch 1986) (family Ambystomatidae), Triturus marmoratus (Blanco and Alberch 1992) (family Salamandridae),
and Salamandrella keyserlingii (Vorobyeva and Hinchliffe
1996) (family Hynobiidae), all of which have pond-type larvae in which limb development proceeds slowly and continues well after hatching (typically, only forelimb buds are
present at hatching).
Marks et al. (see footnote 2) investigated limb development in two species from remotely related genera in the
family Plethodontidae that display direct development and
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Fig. 4. Skeletons of the hand (left) and foot (right) of a young adult (approximately 65 mm SVL) D. ensatus from Jenner, Sonoma
County, California. Bone is outlined and cartilage is stippled. For an explanation of abbreviations see Figs. 2 and 3.
from one member of that family that has an extended period
of intracapsular development and a stream-type larva (see
footnote 2). These plethodontids resemble other salamanders
and differ from frogs and amniotes in showing an early
preaxial dominance in development, with the limb axis extending through digit 2 rather than through digit 4, and in
showing precocious development of the basale commune in
an isolated distal position. However, they differed in having
a less pronounced digital arch and more simultaneous development of distal mesopodial elements. In these respects,
plethodontids displayed more amniote-like and frog-like
characteristics. In amniotes, limbs develop during the embryonic period and they are not used as they are forming. In
frogs, the forelimbs, in particular, are enclosed within the
gill chamber and form slowly, during which time they are
not used. The direct-developing salamanders and those with
extended intracapsular development have similar development. Accordingly, Dicamptodon offers an opportunity to
test the hypothesis (Marks et al., see footnote 2) that features
of limb development in salamanders with pond-type larvae
represent larval adaptation and that species with intracapsular development will show more similarities to frogs and
amniotes with respect to some features of limb development.
The most appropriate taxon for comparative analysis is
the sister taxon of the Dicamptodontidae, the Ambystomatidae (Larson and Dimmick 1993). Dicamptodon lays eggs in
streams, undergoes much intracapsular development, hatches
with both forelimbs and hind limbs relatively well devel-
oped, and has larvae that stay in streams for at least 2 years.
In contrast, A. mexicanum (and most other members of this
speciose genus) lays eggs in ponds, has relatively little intracapsular development, has pond-type larvae, and hatches
with incompletely developed forelimbs and almost no hind
limbs. The larvae of most species of Ambystoma metamorphose within a few months of hatching. Dicamptodon differs
substantially from Ambystoma in the overall degree of limb
development achieved prior to hatching. It also differs in
that its limb buds form small paddles rather than being bulletshaped. However, with respect to details of internal limb development, Dicamptodon resembles A. mexicanum (Shubin
and Alberch 1986). Development of the limb in all salamanders is a series of bifurcation and segmentation events. The
primordia of the proximal element of the limbs (humerus
and femur) bifurcate to connect to paired distal elements (radius and ulna and tibia and fibula, respectively). The
preaxial member of each pair gives rise to a preaxial column, which then segments twice, producing the radiale and
element y in the manus and the tibiale and element y in the
pes. The postaxial member of each pair bifurcates, giving
rise to medial and postaxial columns. The medial column
starts to bifurcate into a centrale and element m, but this
process is aborted and the column essentially segments to
form the definitive centrale, which incorporates element m
(Fig. 5; see also Fig. 1 in Shubin and Wake 1996). The
postaxial column ceases its independent growth without either segmenting or bifurcating and becomes either the ulnare
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Fig. 5. Patterns of connectivity, both primary and secondary, in the development of the limb of diverse salamander species. (A) From
Fig. 2A, a preparation stained with antibodies to type II collagen, showing connectivities. (B) General model of pattern of limb
development in the family Dicamptodontidae, based on data given for Dicamptodon in this paper. (C) General model of the pattern of
limb development in a member of the family Salamandridae, based on Triturus marmoratus (from Blanco and Alberch 1992; Shubin et
al. 1995). (D) General model of the pattern of limb development in a member of the family Ambystomatidae, based on Ambystoma
mexicanum (from Shubin and Alberch 1986; Shubin et al. 1995). pr, prepollex; for an explanation of other abbreviations see Figs. 2
and 3.
or the fibulare. This pattern is essentially identical in Dicamptodon and Ambystoma and appears to be fundamental
to all salamanders that have been studied to date. Another
fundamental feature associated with caudate limb development is the precocious development of digits 1 and 2 and the
associated basale commune in both the forelimb and the
hind limb. Thus, the primary axis of the limb extends
through digit 2, rather than through digit 4 as is typical of
frogs and amniotes (Shubin and Alberch 1986). Furthermore,
there is a distinct digital arch that develops in a preaxial to
postaxial gradient. Where the digital arch approaches the
postaxial column, the last one or two distal carpals or tarsals
form and the last two digits appear. The last digit to appear
is the most postaxial, and as in other salamanders, there are
only four digits on the forelimb. However, Dicamptodon differs from Ambystoma in the formation of a ringlike series of
condensations in the postaxial portion of the manus and pes
(Fig. 5) (a similar arrangement was illustrated for S. keyserlingii by Schmalhausen 1910). Although there is early separation of the basale commune from more basal elements,
later there develops a continual dense matrix out of which
form distal carpal 4 and the fourth digit in the manus and
distal tarsals 4 and 5 and the fourth and fifth digits in the
pes. Accordingly, the digital arch of Dicamptodon is trun-
cated and limited to the basal commune and distal element 3
(as in T. marmoratus; Blanco and Alberch 1992) (Fig. 5).
In some respects, limb development in Dicamptodon is
more similar to that found in plethodontids (Desmognathus
and Bolitoglossa; see footnote 2) than in ambystomatids.
These include the degree of differentiation prior to hatching,
the weak postaxial extension of the digital arch, and the absence of a bullet-shaped external form. These differences
may reflect adaptive changes associated with life-history
evolution. Hatchlings of species with pond-type larvae are
altricial, with tiny or no limb buds. Such limbless larvae survive well. They have paired anterior appendages, balancers,
that function partially as limbs until forelimbs appear (reviewed by Crawford and Wake 1998). By contrast, in species having larvae that hatch in streams, there is high
survival value in having well-developed limbs at the time of
hatching in order that the organisms will be better able to
maintain their position in streams as they move about on the
bottom. Nevertheless, such fundamental salamander features
as preaxial dominance are strongly manifest in Dicamptodon
and appear to have evolved early in the history of the Caudata.
Dicamptodontidae and Ambystomatidae are sister taxa, so
the several differences between them need to be partitioned
as to whether they are derived or ancestral. Certainly, there
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Wake and Shubin
has been homoplasy because ambystomatids resemble hynobiids and salamandrids in several respects, while dicamptodontids resemble plethodontids. We are presently engaged in
a broad comparative study of limb development in the
Caudata (e.g., Shubin and Wake 1996), but the absence of
close outgroups makes assessment of synapomorphies problematic. For example, the transient appearance of element m
in both dicamptodontids and ambystomatids probably represents a retained ancestral trait, but the expression of element
m in occasional adult plethodontids and its association with
distal mesopodials in several unrelated plethodontid lineages
(Wake 1991; Shubin and Wake 1996) represent apomorphic
change and homoplasy. Whether the general pattern of limb
development in dicamptodontids is convergent with that in
other stream-dwelling taxa or represents retention of an ancestral trait is uncertain.
We believe that several of the features found in Dicamptodon as well as in other salamanders are synapomorphies at
the level of the Caudata. These include those traits in which
salamanders differ from anurans and amniotes: preaxial
dominance in development, precocious development of the
basale commune and digits 1 and 2, a limb axis that goes
through digit 2, and a digital arch that grows from preaxial
to postaxial. In addition, all salamanders have a unified
basale commune (an amalgamation of the first two distal elements) in both the manus and the pes, and no salamander
has more than four digits on the forelimb (Shubin et al.
1995; Shubin and Wake 1996). However, while these features are constant, there is reason to believe that some of
them (e.g., preaxial developmental dominance and precocious development of the first two digits) may have become
accentuated by adaptive processes in those species with
pond-type larvae. In such larvae, especially those in the family Salamandridae, the second digit becomes extremely elongated (e.g., Blanco and Alberch 1992) and may play a
sensory role. In larvae of the family Hynobiidae, a curious
filamentous structure that has the external form of a spine is
present between the first and second digits of the manus
(e.g., Vorobyeva and Hinchliffe 1996); it may function in locomotion and maintenance of balance. These specializations
appear after hatching, and whatever function they serve
starts prior to full limb development. Thus, they function as
the limbs are developing, and this may be a proximal selective factor leading to preaxial dominance and precocious
distal development of the basale commune and digits 1 and
2. If these features are synapomorphies of the Caudata, retention of them in the dicamptodontids, in a more subdued
form than in the salamandrids, ambystomatids, and
hynobiids, may represent a recapitulatory aspect of limb development.
The new data on Dicamptodon further buttress the basic
division, between salamanders on the one hand and frogs
and amniotes on the other, that has long been noted and discussed (Shubin and Alberch 1986). However, we do not envision these differences as having the significance they were
given in the past, when they led to such extreme interpretations as the diphyletic origin of tetrapods (Holmgren 1933).
The fact that frogs resemble amniotes in most of the features
of limb development discussed here might be taken as support for these features being symplesiomorphic features of
2065
tetrapods, with the salamanders displaying apomorphic traits.
If one accepts that the metapterygial axis of sarcopterygian
fishes gave rise to the digital arch, then one might argue that
the proximodistal sequence of development of lungfish fins
corresponds to the posterior to anterior pattern of digital
arch expansion in amniotes and frogs, and the salamander
anterior to posterior pattern represents an apomorphy of salamanders. However, the paucity of outgroup taxa that would
permit a proper phylogenetic analysis makes this suggestion
problematic. We further explore the balance between adaptive modifications and phylogenetic effects in a comprehensive review of limb development in Caudata that is in
preparation.
We thank E.D. Brodie III for finding and collecting the
eggs in the field and expending considerable effort to have
them safely delivered to the Berkeley laboratory. The eggs
were maintained in the laboratory and preserved by Andres
Collazo and Andrew Crawford. Rebecca Beroukhim developed a staging table that helped us to make correlations between the two clutches of eggs. D.B.W. was trained in immunohistological methods in the laboratory of James Hanken at
the University of Colorado, Boulder, and he thanks J.
Hanken, D. Moury, and D. Jennings for assistance and instruction and J. Hanken for taking photographs of the immunological preparations. We appreciate discussion with
members of the Wake research group and thank S. Kuchta,
M. García-París, G. Parra, and K. Zamudio for review of the
manuscript. Research in the Shubin and Wake laboratories
has been sponsored by the National Science Foundation.
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newt, Triturus marmoratus. Evolution, 46: 677–687.
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